The present invention relates to amino-modified polysaccharides and more particularly, to methods of generating and using the same, such as in treating viral infections such as AIDS.
HIV is the causative agent of acquired immunodeficiency syndrome (AIDS), a fatal human disease, which has affected numerous individuals worldwide. A vast amount of research is currently being undertaken to find new therapies and new drugs, which may provide some assistance in combating the HIV virus. One approach for drug therapy is to target viral proteins in an attempt to inhibit or halt viral replication. Presently, a triple-drug “cocktail” therapy regimen is the most effective approach to controlling the AIDS virus. This “cocktail” therapy includes a combination of a protease inhibitor called indinavir™ with two reverse-transcriptase inhibitors known as AZT™ and 3TC™. The triple drug therapy results in a decrease in measured levels of virus in both blood and lymphatic tissues. However, a proportion of those treated, whose viruses had developed resistance to one or more of the cocktail's reverse-transcriptase inhibitors as a result of previous treatment, fail to respond. Some studies have projected that the combination therapy can virtually eliminate the AIDS virus within two or three years from those patients who respond to treatment. However, other studies indicate that minimal residual virus is sufficient to cause relapse. In addition, current pharmacologic therapies leave residual HIV genes lurking in cells in the form of latent “provirus.” As long as the cells remain alive, these genes can be transcribed, facilitating virus production. For these reasons, currently available drug therapy must be maintained throughout patient's life, resulting in long term adverse side effects and treatment costs of over $15,000 annually for each patient.
Thus, there is a continuing need to develop additional approaches to controlling HIV infection which can be used alone or in combination with existing therapies.
The process of HIV-1 viral infection involves the interaction of the gp120 Env protein with the CD4 receptor and the CXCR4 and CCR5 chemokine coreceptors on the target cell (reviewed in Berger, E. A. et al., 1999. Annu. Rev. Immunol. 17: 657–700; Cammack, N. 1999. Antivir. Chem. Chemother. 10: 53–62; Choe, H. et al., 1998. Semin. Immunol 10: 249–257; Clapham and McKnight 2001. Br. Med. Bull. 58: 43–59; Zaitseva, M. et al., 2003. Biochim. Biophys. Acta 1614: 51–61). These interactions form a tri-molecular complex (gp120-CD4-chemokine receptor) which stabilizes virus binding and triggers a series of conformational changes in the second Env protein, gp41. These conformational changes lead to lipid mixing and fusion of the cellular and viral membranes, which result in entering of the viral core into the cell (reviewed in J. P. Moore and R. W. Doms, 2003. Proc. Natl. Acad. Sci. U.S.A 100: 10598–10602).
The discovery of virus entry and the consequent understanding the receptor-induced conformational changes in the Env protein and virus-cell fusion, led to the development of entry inhibitors [J. P. Moore and M. Stevenson, 2000. Nat. Rev. Mol. Cell Biol. 1: 40–49; B. M. O'Hara and W. C. Olson, 2002. Curr. Opin. Pharmacol. 2: 523–528]. The overall viral entry process (binding and fusion) can be blocked by a number of compounds. These include siamycin analogues, SPC 3 (a synthetic peptide derived from the V3 domain of gp120), pentafuside (T20, DP178, a synthetic peptide corresponding to amino acid residues 127 to 162 of gp41), the betulinic acid derivative RPR 103611, TAK 779 (a low molecular weight non-peptide CCR5 antagonist) and a number of compounds (T22, T134, ALX40-4C, CGP64222 and AMD 3100) that target the CXCR4 coreceptor (reviewed in Zaitseva, M. et al., 2003. Biochim. Biophys. Acta 1614: 51–61; De Clercq, E. 2002. Med. Res. Rev. 22: 531–565; De Clercq, E. 1999. Drugs R.D. 2: 321–331; Este, J. A. 2003. Curr. Med. Chem. 10: 1617–1632; Baldwin, C. E. et al., 2003. Curr. Med. Chem. 10: 1633–1642). One of these entry inhibitors, T20 (Trimeris, Durham, N.C.), has been approved for clinical use (M. L. Duffalo and C. W. James, 2003. Enfuvirtide: A Novel Agent for the Treatment of HIV-1 Infection. Ann. Pharmacother. 37:1448–1456). However, since the entry inhibitors are targeted against the viral Env protein, which is the most variable protein among primary HIV-1 viruses, the various HIV-1 strains have different sensitivity towards the entry inhibitors. In fact, the extent of strain-to-strain variation is markedly greater for entry inhibitors than it is for reverse transcriptase and protease inhibitors [Moore and Doms (Supra)].
Recently, RNA has been recognized as a target site for therapeutic intervention because of its central role in protein synthesis [Pearson and Prescott (1997) Chem. Biol. 4:409–14]. The advantages of targeting RNA over traditional protein targets include the slower development of drug resistance against small molecules, the high conservation of RNA functional domains and accessibility of RNA functional domains to drugs. The HIV virus, which has rapidly developed resistance to enzyme inhibitors, has thus become an important target for RNA-targeted small molecules. The trans activating region (TAR) RNA and the Rev responsive element both responsible for gene regulation in HIV, have been identified as possible RNA-based targets. The RNA functional domain of TAR binds the cognate peptide Tat, which activates transcription of the HIV genome.
HIV-1 specific ribozymes, antisense RNA, and RNA decoys have also been proposed as potential therapeutic reagents for HIV-1 [Chatterjee, S., et al., Science 258, 1485–1488 (1992); Sullenger, B. A., et al., Cell 63, 601–608 (1990); Ojwang, J. O., et al., PNAS 89, 10802–10806 (1992)]. Some examples are provided in WO92/05195 which discloses molecules that mimic the high-affinity binding site of the native RRE in order to act as competitive inhibitors, thus sequestering free Rev protein and preventing it from interacting with those mRNAs which contain the RRE. These molecules contain a greater number of Rev binding sites than are contained in viral RRE-containing mRNAs.
Additionally, Jensen and co-workers (1995) disclose chemically modified RNA sequences (i.e., containing 5-iodouridine) which bind Rev in vitro with higher affinity than the RRE and which are able to crosslink with Rev at a 1:1 ratio. These are postulated as potential suicide ligands for in vivo disease inhibition, however, non-specific interactions with chemically reactive bases cannot be ruled out under in vivo conditions.
Aminoglycosides have found clinical use as antibacterial agents, owing to their ability to bind 16S RNA of 30S subunit of bacterial ribosomes, which could alter the function of the target RNA [Noller (1991) Annu. Rev. Biochem. 60:191–227]. Aminoglycosides were also shown to interact with a large number of other RNAs including the two essential elements of the HIV genome, the Rev responsive element (RRE) and the transactivation responsive element [(TAR); Zapp (1993) Cell 74:969–978; Mei (1995) Bioorg. Med. Chem. Lett. 5:25755–2760]. For example, the ability of neomycin B to bind at the minor groove of HIV TAR RNA leading to conformational changes in TAR, thus restricting Tat binding to the major groove of TAR-RNA has been independently exhibited by Faber (2000) J. Biol. Chem. 275:20660–6, and Puglisi (1993) Proc. Natl. Acad. USA 90:3680–3684.
NMR studies addressing aminoglycoside antibiotic binding to RNA suggest that rings I and II of the neomycin-class aminoglycosides are sufficient for mediating the specific interaction with the RNA [Fourmy (1998) J. Mol. Biol. 277:347–362]. However, additional rings and amino groups in this class of antibiotics increase RNA binding affinity [Ryu (2002) Biochemistry 41:10499–509].
Arginine- and lysine-rich basic peptides include a common motif of RNA recognition by proteins. Thus, for example, HIV Tat and Rev proteins mediate their interactions with the viral RNAs via an arginine-rich motif [Weeks (1990) Science 249:1281–1285]. Although the dominant contributions of the arginine side-chains may differ between complexes, the ability of the guanidinium groups of the arginine side chains to be involved in the electrostatic interactions, hydrogen bond formation, π—π and stacking interactions make arginine an important moiety for RNA recognition [Cheng (2001) Curr. Opin. Struct. Biol. 11:478–484].
Attempts to mimic the arginine-rich peptides led to the development of novel RNA ligands, which utilize a diverse set of building blocks [Litovchick (1999) FEBS Lett. 445:73–79; Litovchik et al. (2000) Biochemistry 39: 2838–2852]. Arginine-rich RNA-binding peptides and peptidomimetics have provided a good scaffold for RNA-targeted drug design since they are short, conformationally diverse and contact RNA with high affinity and specificity.
The present inventors have recently designed and synthesized aminoglycoside-arginine conjugates (AACs) as potential anti-HIV-1 agents that combine the RNA binding ability of aminoglycosides and the specific binding of arginine moiety to HIV-1 TAR RNA (WO00/39139). AACs are designed to bind HIV TAR RNA and to inhibit trans-activation by Tat protein. AACs are antagonists of the HIV-1 Tat protein basic domain and structurally are peptidomimetic compounds with different aminoglycoside cores and different numbers of arginines [Litovchick (1999) Supra.; Lapidot (2000) Drug Dev. Res. 50:502–515]. Along with inhibition of Tat trans-activation step in HIV life cycle, AACs exert a number of other activities, closely related to Tat antagonism. For example, hexa arginine neomycin B conjugate (NeoR6) inhibits the several functions of extra cellular Tat protein including upregulation of the HIV-1 viral entry co-receptor (CXCR4), increase of viral production, suppression of CD3-induced proliferation of lymphocytes, and upregulation of CD8 rereptor [Litovchick (2001) Biochemistry 40:15612–15623]. It was recently shown that NeoR6 and tri-arginine gentamicyn conjugate (R3G) inhibit binding of HIV particles to cells, probably by blocking the CXCR4 co-receptor [Litovchick (2000) Biochemistry 39:2838–2852; Litovchick (2001) Supra]. This was further substantiated by the finding that NeoR6 competes with the binding of the monoclonal antibody 12G5 to CXCR4, and CXCR4-SDF-1α binding [Litovchick (2001) Supra] and inhibits elevation of intracellular Ca2+ induced by SDF-1α[Cabrera (2002) Antiviral Res. 53:1–8; Cabrera (2000) AIDS Res. Hum. Retroviruses 16:627–634]. Noteworthy is that AACs penetrate a variety of mammalian cells, including neurons and accumulate intracellularly [Litovchick (2001) Supra; and Litovchick (1999) Supra]. In particular, NeoR6 was shown to cross the blood brain barrier when administered systematically thereby penetrating various brain tissues (Catani, M. V. et al., 2003. J Neurochem. 84: 1237–45). All these render AACs multifunctional HIV Tat antagonists and therefore are highly important novel class of anti viral drugs.
The present inventors also found that AACs (e.g., NeoR6 and R3G) are able to elicit inhibition of bacterial RNAse P (Eubank T D, et al., 2002. Inhibition of bacterial RNase P by aminoglycoside-arginine conjugates. FEBS Lett. 511: 107–12) and to a lesser extent, mammalian RNAse P, which inhibition is far more significant than the inhibition elicited by their unconjugated aminoglycoside counterparts. Recently the capacity of several AACs to inhibit translation has also been presented [Carriere (2002) RNA 8:1267–1279].
In view of the ever-expanding roles of AACs in antibacterial and antiviral therapies, it is highly desirable to further elucidate the structural functional relationship of AAC binding to RNA and proteins, in order to design and identify anti viral drugs with improved therapeutic efficacy and reduced cytotoxicity.
While reducing the present invention to practice the present inventors have uncovered a new approach which allows for the synthesis of novel AACs with improved anti-bacterial and anti-viral activities.